Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US. The goal of the research proposed in this R21 project is to identify and develop drugs that block the function of Sonic Hegehog (Shh), a key contributor to pancreatic tumorigenesis. Aberrant expression of Shh is implicated in the biogenesis of human pancreatic cancer. In order to signal correctly, Shh must be modified by attachment of the 16-carbon fatty acid palmitate to its N-terminus. We aim to exploit Shh palmitoylation as a potential Achilles heel by targeting Hhat (Hedgehog acyltransferase), the enzyme that catalyzes attachment of palmitate to Shh. Since palmitoylation is required for Shh function, Hhat inhibitors that block Shh palmitoylation could be developed into novel chemotherapeutics that will be efficacious in the treatment of pancreatic cancer. My laboratory has developed an in vitro Shh palmitoylation assay that is dependent on Hhat activity. The assay has been optimized for High Throughput Screening (HTS) to identify small molecule inhibitors of Hhat. We are nearing completion of the HTS (a total of 80,000 compounds) and estimate selection of 200-300 potential hits for secondary analysis. We now seek funding to identify the inhibitor hits that are the most effective in blocking Shh-driven pancreatic cancer cell growth. Aim 1) To perform secondary screening of the HTS hits to identify novel and effective small molecule inhibitors of Hhat-mediated Shh palmitoylation. Compounds that yielded >70% inhibition and a B-score >12 in the HTS assay will be analyzed in secondary HTS screens. The ability of these Hhat inhibitors to inhibit Shh palmitoylation in normal and pancreatic cancer cells will then be assessed. Aim 2) To identify Hhat inhibitors from the HTS that inhibit proliferation of pancreatic cancer cells. We have shown that shRNA mediated depletion of Hhat inhibits anchorage- dependent and independent growth of human pancreatic cancer cells and also blocks paracrine Shh signaling. These findings validate the choice of Hhat as a defined molecular and therapeutic target. Experiments in Aim 2 will assess the ability of the Hhat inhibitors from the HTS to inhibit proliferation, survival and migration of human pancreatic cancer cells. Three-dimensional Matrigel-based culture systems will be established and the effects of Hhat inhibitors on autocrine and paracrine Shh signaling will be determined. The most effective inhibitors will be developed into lead compounds for further therapeutic development. PUBLIC HEALTH RELEVANCE: Hedgehog signaling has been shown to drive the growth of human pancreatic cancers. The proposed studies will help us develop small molecule inhibitors that block the functioning of Hedgehog proteins in cancer cells. These compounds could potentially be clinically useful as anti-tumor agents to treat human pancreatic cancer.